Pilot-Scale Compound Screening against RNA Editing Identifies Trypanocidal Agents

Abstract

Most mitochondrial messenger RNAs in trypanosomatid pathogens undergo a unique type of posttranscriptional modification involving insertion and/or deletion of uridylates. This process, RNA editing, is catalyzed by a multiprotein complex (~1.6 MDa), the editosome. Knockdown of core editosome proteins compromises mitochondrial function and, ultimately, parasite viability. Hence, because the editosome is restricted to trypanosomatids, it serves as a unique drug target in these pathogens. Currently, there is a lack of editosome inhibitors for antitrypanosomatid drug development or that could serve as unique tools for perturbing and characterizing editosome interactions or RNA editing reaction stages. Here, we screened a library of pharmacologically active compounds (LOPAC 1280 ) using high-throughput screening to identify RNA editing inhibitors. We report that aurintricarboxylic acid, mitoxantrone, PPNDS, and NF449 are potent inhibitors of deletion RNA editing (IC 50 range, 1-5 µM). However, none of these compounds could specifically inhibit the catalytic steps of RNA editing. Mitoxantrone blocked editing by inducing RNA-protein aggregates, whereas the other three compounds interfered with editosome-RNA interactions to varying extents. Furthermore, NF449, a suramin analogue, was effective at killing Trypanosoma brucei in vitro. Thus, new tools for editosome characterization and downstream RNA editing inhibitor have been identified.

Structure and intrinsic disorder of the proteins of theTrypanosoma brucei editosome

“…The different diseases represent severe problems mostly in tropical and subtropical regions of the globe and are responsible for about one million deaths per year [1]. A promising drug target for the different diseases is the unique mRNA editing process that takes place within the mitochondria of kinetoplastids [2,3]. Mitochondrial pre-mRNAs in these organisms undergo a multistep RNA editing reaction, which leads to the maturation of mRNAs and by introducing variation, it may contribute to the generation of protein diversity [4,5].…”

A novel high-throughput activity assay for theTrypanosoma bruceieditosome enzyme REL1 and other RNA ligases

“…Most likely it acts on several T. brucei pathways and the exact mode of action is still not entirely clear ( 42 , 44 , 45 ). Suramin and its analogue NF 023 have previously been identified as inhibitor of RNA editing in vitro ( 32 , 46 ). One study suggested that NF 023 and suramin ‘are either acting at a step following the endonuclease cleavage or alternatively having global effects on the editing complex’ ( 32 ).…”

Repurposing approved drugs on the pathway to novel therapies

“…Many chemotherapy drugs that require infusion, such as the bright blue compound mitroxantrone, an agent which has a variety of activities in cells, have limited solubility and may induce nonspecific aggregation of the protein or assay reagents in high throughput screening . A good deal of time can be lost to these nonspecific aggregators, which can have misleading activity in a variety of different assays at low micromolar concentrations . Mitroxantrone even turned up in a preliminary screen to identify inhibitors of uridylyation of the peptide linked to the genome, VPg, to VPgpU by the coxsackie virus A24 polymerase in the author's group.…”

“…Thus, the approximately 4000 compounds used in the clinic are now being reinvestigated, to see what other treatments they could provide for difficult to treat conditions. Searching approved drugs for new purposes has been aided by commercially available compilations of drugs approved by the FDA, such as the Prestwick Chemical Library, the Library of Pharmacologically Active Compounds (LOPAC1280) and others, or specific libraries assembled for various purposes, such as those targeting RNA‐protein interactions or kinases. The “Drug Repurposing Hub” (http://www.broadinstitute.org/repurposing), consisting of both a reliable physical source for approved drugs, as well as a virtual library describing their characteristics, will certainly help to identify new uses for the existing pantheon .…”